Novel notch-origin polypeptides and biomarkers and reagents using the same

ABSTRACT

It is intended to provide extracellular markers whereby Notch signal transduction can be detected. Polypeptides (Nβ), which are novel peptides originating in Notch protein and released form cells in the step of the nuclear migration of NICH (Notch intracellular cytoplasmic domain) due to the extracellular digestion and the subsequent protein digestion in the membrane during a series of the Notch protein digestion, are referred to as markers. These peptides (Nβ) are released from the cells in proportion to the Notch signal depending on presenilin. By detecting these peptides, the Notch signal transduction, cell differentiation, cell tumorigenesis, apoptosis, Alzheimer&#39;s disease, etc. can be monitored.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 10/521,691,filed Aug. 31, 2005, which is a U.S. National Stage application ofPCT/JP03/09059, filed Jul. 17, 2003, which applications are incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates to novel polypeptides derived from novelintramembranous endoproteolysis of Notch proteins (hereinafter alsoreferred to collectively as “Notch”) and to biomarkers and reagentsusing the same. In the description of the present invention, thefollowing abbreviations are used for cleavage sites of Notch: S1 forSite-1, S2 for Site-2, S3 for Site-3, and S4 for Site-4. As will bedescribed later, Site-4 (S4) is a novel intramembranous cleavage sitediscovered by the inventors of the present invention.

BACKGROUND ART

Notch is a type I transmembrane protein present on a cell surface. Itcontains a repeated EGF-like domain in its extracellular domain and NICD(Notch Intracellular Cytoplasmic Domain), which is a transcriptionfactor containing an ankyrin repeated domain, in its intracellulardomain. It has been known that Notch plays a role in intracellularsignaling relating to cell differentiation. For example, in thedevelopmental process of a cranial nerve system, some of the cellsderived from ectoderm differentiate into neuronal precursor cells (stemcells) and further into nerve cells or glial cells, during whichintracellular signaling via Notch is important. The mechanism of theintracellular signaling via Notch is as follows. First, Notch isexpressed as a receptor on a Notch signal-receiving cell. During thetransport to the cell surface, the Notch undergoes the cleavage at theextracellular domain (S1) by a protease such as furin, and the two Notchfragments resulting from the S1 cleavage are held together through anS—S bond on the cell surface. Next, when a Notch signal-sending cell ispresent near the Notch signal-receiving cell, a Notch ligand (e.g.,Delta, Serrate, or Lag-2, belonging to a DSL family) is expressed on thesurface of the Notch signal-sending cell. Under these two conditions,the Notch ligand interacts with the Notch receptor on the cell surface,whereby sequential proteolytic events are induced to trigger signaltransduction. More specifically, the Notch is cleaved at a site (S2)close to the cell surface, which triggers the cleavage at a site (S3)that is either inside the cell membrane or in close proximity to thecell membrane inside the cell. NICD, which is the intracellular domainof the Notch resulting from the S3 cleavage, is released to anintracellular space and translocates to the nucleus, where it binds to aCSL family (CPB, SuH, or Lag-1; transcription factor) to regulate thetranscription of target genes. Presenilin, which is associated withAlzheimer's disease, is involved in the S3 cleavage.

As described above, Notch plays an extremely important role inintracellular signaling for cell differentiation. Moreover, recentstudies have revealed that Notch is involved not only in thedifferentiation of a cranial nerve system as described above but also incell tumorigenesis, apoptosis, Alzheimer's disease, etc., which causesNotch to become a focus of attention (see Okochi et al., “Biology ofAlzheimer's disease and presenilin”, Bunshi Seishin Igaku, Vol. 1, No.3, 2001; Kageyama et al., “Notch pathway in neural development”,Tanpakushitsu Kakusan Koso, Vol. 45, No. 3, 2000; and Brian et al., “Acarboxy-terminal deletion mutant of Notch 1 accelerates lymphoidoncogenesis in E2A-PBX1 transgenic mice”, Blood, Vol. 96, No. 5, 2000Sep. 1, pp 1906-1913). Therefore, the detection of Notch signaltransduction is extremely important for research and diagnosis of celldifferentiation, cell tumorigensis, apoptosis, Alzheimer's disease,etc., and the earlier possible establishment of the technology fordetecting Notch signal transduction is being demanded.

DISCLOSURE OF INVENTION

Therefore, with the foregoing in mind, it is an object of the presentinvention to provide a substance that can serve as an extracellularsecreted marker for detecting Notch signal transduction.

The inventors of the present invention hypothesized that, during aseries of proteolytic events of Notch, a polypeptide remaining in a cellmembrane is released to an extracellular space as a result of thecleavage occurring at S3, and decided to examine this hypothesis. Thisis because, if the polypeptide remaining in the cell membrane isreleased to an extracellular space, it can serve as a marker for Notchsignal transduction. Through a series of studies on Notch signaltransduction, the inventors of the present invention found out that afourth cleavage occurs at a site (in the transmembrane domain) differentfrom the S3 cleavage site and a polypeptide resulting from this fourthcleavage is released to an extracellular space. Based on this finding,the inventors arrived at the present invention.

That is, the novel polypeptide according to the present invention is apolypeptide derived from a Notch protein. In a series of proteolyticevents of the Notch protein, the polypeptide is released to anextracellular space when NICD (Notch intracellular cytoplasmic domain)translocates to a nucleus as a result of the intramembranousendoproteolysis that occurs subsequent to the extracellular proteolysis.This polypeptide can be detected by using an antibody or the like, andthus can be used as a marker for detecting Notch signal transduction.Furthermore, since Notch signal transduction is involved in celldifferentiation, cell tumorigensis, Alzheimer's disease, apoptosis,etc., the novel polypeptide according to the present invention also canbe used as a marker for detecting them. Moreover, as will be describedlater, there are several types of novel polypeptide according to thepresent invention with their C-termini being different from each other.Hereinafter, the novel polypeptide according to the present invention isreferred to also as “Notch-β (Nβ)”. Also, the above-describedintramembranous endoproteolysis is not limited to that occurring in acell membrane but includes that occurring in an organelle membrane.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic illustration of structures of NΔE, FLAG-NEXT(F-NEXT), and NICD. FIGS. 1B and 1C are electrophoretograms showing anexample of the production of FLAG-tagged novel polypeptides (Nβs)according to the present invention.

FIGS. 2A and 2B are electrophoretograms showing an example of theproduction of novel polypeptides (Nβs) according to the presentinvention.

FIG. 3A is a chart showing the result of mass spectroscopy with regardto a group of novel polypeptides according to the present invention.

FIG. 3B shows a major site of a novel cleavage (S4 cleavage) of a Notchprotein and major cleavage sites of an Alzheimer's disease (3-amyloidprecursor protein (hβAPP).

FIG. 4A shows an example of amino acid sequences of the novelpolypeptides as a principle part of the present invention. FIG. 4B is aview showing the comparison between intramembranous amino acid sequencesof Notch-1 to Notch-4 and that of hβAPP.

FIGS. 5A and 5B are electrophoretograms showing an example of the effectof inhibition of presenilin (PS) function upon extracellular release ofnovel polypeptides (Nβs) according to the present invention.

FIG. 6A is a chart showing the result of mass spectroscopy, which showsan example of the effect of Alzheimer's disease pathogenic presenilinmutants upon Nβ release. FIG. 6B shows Nβ species whose secretion isrelatively increased by the effect of Alzheimer's disease pathogenicpresenilin mutants. FIG. 6C shows the result of a semiquantitativeanalysis of the relative increase of their secretion.

FIG. 7 is a schematic illustration of an example of extracellularrelease of novel polypeptides (Nβs) according to the present inventionand illustrates the C-terminus of the released peptide is changed byAlzheimer's disease pathogenic presenilin mutants.

FIG. 8A illustrates how cleavages occur in transmembrane domains ofNotch-1 and βAPP. FIG. 8B is a schema specifically illustrating F-NEXTV1744G and F-NEXT V1744L mutants. FIG. 8C is an electrophoretogramshowing an example of inhibition of NICD production caused by mutatingV1744. FIG. 8D is an electrophoretogram showing an example of F-Nβsecretion in the corresponding cell culture media. FIG. 8E shows theresult of the measurement of S3 and S4 cleavage efficiencies in thecells.

FIGS. 9A, 9B, and 9C are charts showing the result of mass spectroscopywith regard to F-Nβ peptides released from wild-type F-NEXT, F-NEXTV1744G mutant, and F-NEXT V1744L mutant, respectively.

FIG. 10A is a schema specifically illustrating a S4 cleavage site mutantprepared in an example of the present invention. FIGS. 10B and 10C areexamples of electrophretograms showing molecular weights of F-Nβsreleased from wild-type F-NEXT, F-NEXT G1730-1733 mutant, and F-NEXTL1730-1733 mutant, respectively. FIG. 10D shows the result of themeasurement of S3 and S4 cleavage efficiencies in the cells.

FIGS. 11A and 11B are charts showing the result of mass spectroscopywith regard to F-Nβ peptides released from F-NEXT G1730-1733 mutant andF-NEXT L1730-1733 mutant, respectively.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described further in detail.

A polypeptide according to the present invention is released to anextracellular space in proportion to Notch signal transduction. Besides,novel proteolysis that occurs immediately before the release of thepolypeptide to the extracellular space is presenilin dependent, andinhibition of the presenilin function causes a decrease in the releaseof the polypeptide of the present invention.

The novel polypeptide according to the present invention is produced andreleased as a result of the proteolysis (S4 cleavage) of a Notch proteinthat occurs simultaneously with or either before or after theproteolysis of the Notch protein at a S3 cleavage site. The proteolysis(S4 cleavage) occurs on a N-terminal side with respect to the S3cleavage site in a transmembrane domain of the Notch protein.

The novel polypeptide (Nβ) according to the present invention is apolypeptide including an amino acid sequence selected from SEQ ID NOS: 1to 18. In these SEQ ID NOS: 1 to 18, SEQ ID NOS: 1 to 9 represent murineamino acid sequences, while SEQ ID NOS: 10 to 18 represent human aminoacid sequences. In the amino acid sequences represented by the SEQ IDNOS: 1 to 18, one or several of the amino acids may be deleted,substituted, or inserted. Polypeptides represented by such amino acidsequences also are derived from Notch proteins, and are released to anextracellular space when NICD translocates to a nucleus as a result ofintramembranous endoproteolysis that occurs subsequent to extracellularproteolysis in a series of proteolytic events of the Notch proteins.These polypeptides also are released to an extracellular space inproportion to a Notch signal in a presenilin-dependent manner. It is tobe noted that the novel polypeptide according to the present inventionmay be derived from a living organism or may be synthesizedartificially. The living organism is not limited to a particular type,and may be, for instance, a human, a mouse, a rat, a rabbit, a goat, aswine, a bovine, a drosophila, or a nematode. Also, the type of tissueor cell from which the novel polypeptide of the present invention isderived is not particularly limited. More specifically, somatic cellsand tissues, such as nerve, marrow, and cancer cells and tissues, may bethe source of the polypeptide of the present invention, regardless ofwhether undifferentiated or differentiated.

A biomarker according to the present invention contains theabove-described polypeptide of the present invention. The biomarker ofthe present invention can be used for detecting Notch signaltransduction, cell differentiation, tumor, apoptosis, Alzheimer'sdisease, or the like. The biomarker of the present invention further maycontain other components, or alternatively, it may be the novelpolypeptide itself (i.e., the biomarker may contain the novelpolypeptide alone). This biomarker can be detected using a reagentcontaining an antibody that can recognize the novel polypeptide. Theantibody that can recognize the novel polypeptide can be prepared by anordinary method, and may be a monoclonal antibody or a polyclonalantibody. In addition to the antibody that can recognize the novelpolypeptide, the reagent further may contain a labeled antibody againstthis antibody or a labeled antibody that can recognize the novelpolypeptide. The labeling can be achieved, for example, by using afluorescent substance, an enzyme (e.g., an enzyme that acts on asubstrate that develops color when reacting with the enzyme), aradioactive substance, or a carrier such as agarose.

A gene according to the present invention is a gene encoding the novelpolypeptide of the present invention, and may be DNA or RNA. A vectoraccording to the present invention is a vector containing theabove-described gene, and a transformant according to the presentinvention is a transformant transformed with the above-described vector.

Next, an example of the extracellular release of the novel polypeptideaccording to the present invention will be described with reference tothe left region of FIG. 7. It is to be noted that the right region ofFIG. 7 shows an example of the extracellular release of amyloid-β (Aβ)in Alzheimer's disease. As shown in the left region of FIG. 7, the aminoterminus of NEXT (Notch Extracellular Truncation) is produced as aresult of extracellular cleavage by TACE (TNFβ-Converting Enzyme). TheNEXT resulting from the S2 cleavage then undergoes S3 cleavage, and NICDresulting from the S3 cleavage translocates to the nucleus. Cleavage atS4 (the fourth cleavage site of Notch newly discovered by the inventorsof the present invention) occurs simultaneously with or either before orafter the S3 cleavage, so that Nβ (a novel polypeptide according to thepresent invention) is released to an extracellular space.

Next, an example of C-terminus amino acid sequences of novelpolypeptides of the present invention will be described with referenceto FIG. 4B. FIG. 4B shows sequences near the C-termini of Nβs orfragments released to an extracellular space with regard to 4 types ofmurine Notch (mNotch-1 to mNotch-4), 4 types of human Notch (hNotch-1 tohNotch-4), and hβAPP. As shown in FIG. 4B, the major S4 cleavage siteresides a few amino acid residues closer to the N-terminus with respectto the center of putative transmembrane domain (TM) (indicated by thetriangular arrowhead on the left in the drawing). Furthermore, as shownin FIG. 4B, amino acid sequences around the major cleavage site are notconserved in mNotch-1 to mNotch-4, though valine 1743 as the S3 cleavagesite is conserved (indicated by the triangular arrowhead on the right inthe drawing). Thus, the S4 cleavage site is characterized by itsdiversity, unlike the S3 cleavage. It is speculated that this diversitymight reflect the peculiarity of the mechanism by which S4 secretaserecognizes the sequence of the cleavage site.

EXAMPLES

Hereinafter, the present invention will be described by way of examples.Reagents, materials, and experimental procedures used in the respectiveexamples are as follows.

(Reagent)

A γ-Secretase inhibitor,[(2R,4R,5S)-2-Benzyl-5-(Boc-amino)-4-hydroxy-6-phenyl-hexanoyl]-Leu-Phe-NH2,was purchased from Bachem.

(Plasmids)

cDNAs encoding Notch ΔE-M1727V (NΔE) and NICD with C-terminal 6× c-myctag inserted in pcDNA3 hygro were prepared in the manner described inSchroeter et al. (Schroeter, E. H., Kisslinger, J. A., Kopan, R. (1998),“Notch-1 signalling requires ligand-induced proteolytic release ofintracellular domain”, Nature, 393, 382-386). The cDNAs were gift fromDr. R. Kopan. N-terminally FLAG-tagged NEXT, i.e., FLAG-NEXT (F-NEXT),was prepared by 2-step site-directed mutagenesis. In the first step,F-NEXT M1727V was produced using the ExSite PCR-Based Site-DirectedMutagenesis Kit (Stratagene). NΔE was used as a template, and thefollowing two primers 1 and 2 (SEQ ID NO: 19 and SEQ ID NO: 20) wereprepared.

Primer 1: 5-P-ATCGTCGTCCTTGTAGTCTCTCAAGCCTCTTGCGCCGAGCGCGGGCAGCAGCGTTAG-3′ Primer 2:5-P-GACAAGATGGTGATGAAGAGTGAGCCGGTGGAGCCTCCGCTGCCCT CGCAGCTG-3′

In the second step, F-NEXT was prepared by site-directed mutagenesisusing Quick Change Site-Directed Mutagenesis Kit (Stratagene). TheF-NEXT M1727V was used as a template, and the following two primers 3and 4 (SEQ ID NO: 21 and SEQ ID NO: 22) were prepared.

Primer 3: 5-CCTCGCAGCTGCACCTCATGTACGTGGCAGCG-3′ Primer 4:5-CGCTGCCACGTACATGAGGTGCAGCTGCGAGG-3′

Each mutant was sequenced to verify successful mutagenesis.

(Antibodies)

The polyclonal antibody (L652) is an antibody against a polypeptide withthe amino acid sequence from V 1722 to G 1743 of human Notch-1 (i.e.,the sequence between S2 and S3). The antibody (L652) was produced in thefollowing manner. First, the above-described polypeptide serving as anantigen was provided. This polypeptide is characterized in that itcontains a lot of hydrophobic amino acids. On this account, the antibodywas produced in the same manner as that used for producing an antibodyagainst the Alzheimer's disease amyloid β-protein. More specifically,the antibody was produced in the following manner. The polypeptide wasdissolved in water directly without being conjugated with any carrierprotein. After addition of the same volume of 2× phosphate buffer, thepolypeptide was emulsified with adjuvant and injected into rabbits(Wild-Bode, C., Yamazaki, T., Capell, A., Leimer, U., Steiner, H.,Ihara, Y., Haass, C. (1997), “Intracellular generation and accumulationof amyloid beta-peptide terminating at amino acid 42”, J Biol Chem 272,16085-16088). A monoclonal antibody (9E10) against c-myc and a reagent(M2-agarose) in which a monoclonal antibody against FLAG is covalentlybound to agarose were obtained commercially.

(Cell Cultures and Cell Lines)

Human embryonic kidney 293 (K293), N2a and COS cells were cultured inDMEM supplemented with 10% fetal bovine serum, 1%penicillin/streptomycin, and 200 μg/ml zeocin (to select for PS1expression), and/or 100 μg/ml hygromycin (to select for NΔE and F-NEXTexpression). The K293 can stably express wild-type PS1, PS1 L286V, orPS1 D385N (Okochi et al, 2000, Kulic et al, 2000, Wolfe et al., 1999).The transfection with NΔE or F-NEXT was performed by means of a productnamed Lipofectamine 2000 (Invitrogen).

(Pulse-Chase)

To determine NΔE N-terminal fragment (NTF: Nβ) release from NΔEexpressing cells, K293 cells stably transfected with NΔE or NICD weregrown to confluence in a 10 cm dish. The cells were then metabolicallypulse-labeled for 2 hours with 300 μCi [³H] amino acids (tritiated aminoacid mixture, Amersham) in Earle's Balanced Salt Solution supplementedwith MEM Vitamine Solution (Gibco) and several cold amino acids,followed by a 6-hour chase by 10% FCS/DMEM. To examine Nβ release, cellsexpressing F-NEXT were, at first, starved of methinine for 40 min withmethionine-free media and then pulse-labeled for 1 hour with 400 μCi[³⁵S] amino acid mixture (Redivue Promix, Amersham) in methionine-freeDMEM, followed by chasing for various time periods with the chase mediacontaining 10% FCS/DMEM supplemented with excess cold methionine.

(Immunoprecipitation/SDS-PAGE)

At the End of the Respective Chase Periods, the Media were Collected andput on ice immediately, followed by centrifugation at 3000×g to excludecell debris. Next, a protease inhibitor cocktail (1:1000; Sigma) and0.025% of sodium azide were added. The thus-obtained samples wereimmunoprecipitated with L652 or M2-agarose (Sigma) overnight and thenwashed three times with RIPA buffer containing 0.1% SDS, 0.5%deoxycholic acid, and 1% TritonX-100, followed by SDS-PAGE usingTris-Tricine 10% to 20% gradient gel (Invitrogen). The cells werescraped in ice-cold PBS, and then harvested by means of 1500×gcentrifugation, followed by lysation with 100 μl of 10×RIPA. 900 μl ofPBS with a protease inhibitor mix (1:500; Sigma) was then added to thelysed cells. The insoluble fraction was separated by 15000×gcentrifugation and the resultant supernatant was used forimmunoprecipitation. The samples for immunoprecipitation were pretreatedby protein A sepharose (Sigma) and immunoprecipitated with 9E10 or M2agarose. Next, the washed protein samples were separated by 8% orTris-Tricine SDS-PAGE. After fixation, the gel was shaken in AmplifyFluorographic Reagent (Amersham), dried, and autoradiographed.

(Immunoprecipitation/MALDI-TOF MS Analysis)

After cells stably expressing the F-NEXT and their derivatives weregrown to confluence in a 20 cm dish, the culture media were replacedwith fresh 10% FCS/DMEM. After the cells with the fresh conditionedmedia were cultured for 3 hours in a CO₂ incubator, the culture mediawere collected and immediately put on ice and centrifuged to eliminatecell debris. After supplementation with a protease inhibitor mix(1:1000) and 0.025% sodium azide, the media were immunoprecipitated withM2-agarose for 4 hours at 4° C. The samples were then washed three timesfor 10 min at 4° C. with an MS wash buffer containing 0.1%n-octylglucoside, 140 mM NaCl, 10 mM Tris (pH 8.0), and 0.025% sodiumazide. The samples were then washed once again with 10 mM Tris (pH 8.0)containing 0.025% sodium azide. Peptides bound to the resultantprecipitates were eluted with TFA/Acetonitrile/Water(TFA:acetonitrile:water=1:20:20) saturated with α-cyano-4 hydroxycinnamic acid. The solubilized samples were dried on a stainless plateand subjected to a MALDI-TOF MS analysis. MS peaks were calibrated usingangiotensin (Sigma) and insulin β-chain (Sigma).

Example 1 Detection of N-Terminal Fragment (NTF; F-Nβ) of FLAG-NEXT(F-NEXT) in Culture Media

FIG. 1A is a schematic illustration of structures of NΔE, NICD, andF-NEXT. As shown in FIG. 1A, in F-NEXT, a signal peptide and also a FLAGsequence and two methionines subsequent to the signal peptide areinserted into the N-terminus of NEXT. The 1727th amino acid residue wasnot mutated in the F-NEXT. However, in NΔE (murine Notch-1 (mNotch-1)),methionine 1727 was artificially mutated to valine, as indicated by theinverse triangle in FIG. 1A (Schroeter, E. H., Kisslinger, J. A., Kopan,R. (1998), “Notch-1 signalling requires ligand-induced proteolyticrelease of intracellular domain”, Nature, 393, 382-386). The triangulararrowhead indicates a S3 cleavage site.

Cells stably expressing NΔE or F-NEXT were pulse-labeled for 1 hour with[³⁵S] and chased for the time period indicated in FIG. 1B. The resultantcell lysates were immunoprecipitated with 9E10 and analyzed by 8%SDS-PAGE. As shown in the upper panel of FIG. 1B, proteolysis of NΔE(the middle region of the panel) and F-NEXT (the right region of thepanel) was observed after a 2-hour chase, which resulted in NICD bandsmigrating faster than those of NΔE and F-NEXT. With regard to the NICDproduction efficiency, there was no difference between the cellsexpressing NΔE and the cells expressing F-NEXT.

Next, the culture media were immunoprecipitated with M2-agarose andanalyzed by 8% SDS-PAGE. As shown in the lower panel of FIG. 1B, a bandof F-Nβs (an aggregate of novel polypeptide groups according to thepresent invention) of about 4 kDa was identified only in the 2-hourchased media of the cells stably expressing F-NEXT. The result indicatesan entirely new finding that, during the NICD production, an aminoterminal fragment on the side opposite to the NICD is secreted into anextracellular space.

F-NEXT expressing cells were pulse-labeled with [³⁵S] for 1 hour andchased for the time periods indicated in FIG. 1C. F-Nβs in the media andthe lysates were examined by the above-described experimentalprocedures. As shown FIG. 1C, accumulation of F-Nβs (an aggregate ofnovel polypeptide groups according to the present invention) inaccordance with the extension of chase period was observed in the media,but was hardly detectable in the cell lysates. However, with longerexposure of a film when taking a picture of electrophoresis gel, a F-Nβband with the same molecular weight (hereinafter referred to as “MW”) asin the media was also detectable in the lysates (data not shown).

The results shown in FIGS. 1B and 1C were reproduced when F-NEXT M1727Vmutant was used or when CHO, COS, and N2a were used as the expressingcells (data not shown).

Example 2 Detection of N-Terminal Fragment (NTF: Nβ) of NΔA in CultureMedia

K293 cells stably expressing NΔE or NICD were pulse-labeled with [³H]for 2 hours and chased for 6 hours. Chased media and cell lysates wereimmunoprecipitated with an antibody L652 against NΔE, and thethus-obtained samples were separated by Tris-Tricine SDS-PAGE. As shownin FIG. 2A, a NΔE NTF band (indicated by the triangular arrowhead) of MW3 to 4 kDa was detected in the culture media of the NΔE cells, but notfrom the culture media or cell lysate of the NICD cells. Thus, it wasconsidered that the band shown in FIG. 2A was of wild-type Nβs that werenot FLAG-tagged.

The same media and lysates as in the above were immunoprecipitated withan anti-c-myc antibody (9E10). As shown in the lower panel of in FIG.2B, about 100 kDa bands of NΔE and NICD were detected in the lysates(indicated by the triangular arrowhead), but not in the media. Thisresult suggests that NΔE and NICD were expressed in the respective cellsat substantially the same rate.

Example 3 Identification of C-Termini of Nβs Released to Culture Media

FIG. 3B is a schematic illustration of intramembranous cleavage ofmurine Notch-1 (mNotch-1) and human βAPP (hβAPP). As a result of theintramembranous cleavage of mNotch-1, NICD and Nβ are produced. In thepresent example, Nβ secretion and a novel cleavage site at theC-terminus of Nβ were confirmed. On the other hand, as a result of theintramembranous cleavage of hβAPP, an intracellular fragment CTFγ50(Sastre, M., Steiner, H., Fuchs, K., Capell, A., Multhaup, G., Condron,M. M., Teplow, D. B., Haass, C. (2001), “Presenilin-dependentgamma-secretase processing of beta-amyloid precursor protein at a sitecorresponding to the S3 cleavage of Notch”, EMBO Rep. 2, 835-841.) andseveral types of Aβ fragments are produced.

Culture media of cells stably expressing F-NEXT were immunoprecipitatedwith M2-agarose, and MW of Nβs were analyzed by means of MALDI-TOF MSaccording to the above-described experimental procedures. The result isshown in the large graph shown in FIG. 3A. As shown in the graph,multiple peaks were observed around MW 4000, but no significant peaks ofMW more than 4500 were identified. The small graph shown in FIG. 3Ashows the details of the peaks from MW 3000 to 4500. The same majorpeaks were identified when CHO, COS and N2a were used as host cells(data not shown). These peaks also were identified when transfected withF-NEXT M1727V mutant (data not shown).

FIG. 4A shows a list of amino acid sequences of Nβs corresponding to theMALDI-TOF MS peaks shown in the small graph of FIG. 3A. The C-terminusof the major Nβ species was alanine 1731. Bold letters indicate an aminoacid sequence of the major peak. As shown in FIG. 4A, no peaks of MWaround 5060, corresponding to a S3 cleavage site, were identified. Fromthese results, it can be concluded that Nβs are released to anextracellular space and the cleavage site of the proteolysis occurringjust before the Nβ release is a novel fourth cleavage site (S4) that isdifferent from the conventionally reported three cleavage sites (S1, S2,and S3).

FIG. 4B shows a list of amino acid sequences of transmembrane domains ofhuman (h) and murine (m) Notch-1 to Notch-4. S1, S2, and S3 cleavagesare phenomena common to Notch-1 to Notch-4, and they serve as a commonsignal transduction mechanism through which Notch proteins, whatevertheir species, achieve signal transduction. From these facts, it isspeculated that S4 cleavage also is a phenomenon common to Notchproteins of all types. As shown in FIG. 4B, the S4 cleavage site isconserved partially, similarly to the S3 cleavage site. From this fact,it is speculated that S4 cleavage is a phenomenon common to Notch-1 toNotch-4.

Example 4 Confirmation of Presenilin (PS) Function Dependence ofExtracellular Release of Nβ

Cells expressing wild-type PS1 or PS1 D385N that is a PS1 dominantnegative mutant obtained by artificially causing loss of presenilinfunction were stably transfected with F-NEXT. An hour pulse with [³⁵S]and then a 2-hour chase were performed, and the resulting culture mediaand lysates were analyzed to determine an Nβ release level from thecells expressing both the PS1 derivative and F-NEXT at the same time.First, the chased media were immunoprecipitated with M2-agarose todetect Nβ release. As shown in the upper panel of FIG. 5A, Nβ releasefrom the PS1 D385N expressing cells decreased drastically as comparedwith the case of the wild-type PS1 expressing cells. That is, it wasconfirmed that the S4 cleavage efficiency decreases drastically in thecells expressing the mutant obtained by artificially causing loss ofpresenilin function. Also, the lysates collected at the same time withthe culture media were immunoprecipitated with 9E10. As shown in thelower panel of FIG. 5A, NICD band after the 2-hour chase was hardlyvisible in the PS1 D385N expressing cells. That is, the report that theS3 cleavage efficiency decreases drastically in the cells expressing themutant obtained by artificially causing loss of presenilin function wasreproduced at the same time.

Next, cells stably expressing F-NEXT were pulse-labeled for 1 hour andchased for 2 hours with or without a γ-secretase inhibitor (L685,458)that is designed to bind the active center of presenilin. Morespecifically, 1 μM of L685,458 was added to the culture media 2 hoursbefore methionine starvation. During the pulse-chase period, everymedium used contained the same concentration of L685,458. The chasedmedia were immunoprecipitated with M2-agarose to detect release. Asshown in the upper panel of FIG. 5B, Nβ release from the cells treatedwith the γ-secretase inhibitor decreased drastically. Also, thecorresponding lysates were immunoprecipitated with 9E10. As shown in thelower panel of FIG. 5B, the NICD band after the 2-hour chase period washardly visible due to inhibition of S3 cleavage. From these results, itcan be said that the Nβ release to an extracellular space is caused bypresenilin-dependent proteolysis, and hence, inhibition of thepresenilin function results in the inhibition of S4 cleavage and Nβrelease that occurs subsequent to the S4 cleavage.

Example 5 Effect of Presenilin (PS) Mutant Associated with FamilialAlzheimer's Disease (FAD) Upon S4 Cleavage

Heretofore, various studies have been made on PS mutation associatedwith FAD, and an increase in Aβ secretion has been confirmed in everytype of FAD pathogenic PS mutant. In the present example, it wasconfirmed that PS dependent S4 proteolysis also relates to PS mutationassociated with FAD.

K293 cells expressing wild-type (wt) PS1 or PS1 mutants associated withFAD, namely, PS1 C92S, PS1 L166P, and PS1 L286V, were stably transfectedwith F-NEXT. Then, the culture media of the cells expressing PS1derivatives and F-NEXT were analyzed by MALDI-TOF MS, in order toexamine the change in C-termini of F-Nβs. As shown in FIG. 6A, incontrast to the cells expressing wild-type PS1, characteristic change ina proteolysis pattern of C-termini of Nβs was observed in the cellsexpressing PS1 mutations associated with FAD. In particular, the cellsexpressing the PS1 L166P mutation causing a significant increase in Aβ42production demonstrated a tendency to elongate F-Nβ peptides, and anincrease in the production of F-Nβ species (F-Nβ 1733 and F-Nβ 1735)that were longer than F-Nβ1731 by 2 and 4 amino acid residues,respectively, was confirmed (see FIG. 6B). Furthermore, as shown in FIG.6A, an increase in F-Nβ 1734 level was observed in the PS1 C92S cells,whereas an increase in F-Nβ 1735 level and a decrease in F-Nβ 1734 levelwere observed in the PS1 L286V cells. These results demonstrate that FADpathogenic mutations affects a pattern of the S4 cleavage site so thatthe S4 cleavage site tends to shift toward the C-terminal side, therebycausing elongation of released peptides. Similarly to Aβ42, theaggressive PS1 L166P mutation affects the length of F-Nβs mostsignificantly. It has been known that PS1 L166P mutation causes FADduring the young adult years. These effects were not specific to K293cells, and the same effects of the PS mutations associated with FAD alsowere confirmed when using Neuro 2a cells (data not shown). Therefore, itcan be said that every type of FAD pathogenic mutation affects theC-terminus of F-Nβ (see FIG. 6C).

Example 6 Effect of Proteolysis at S3 Upon Efficiency of Proteolysis atS4

In order to examine the correlation between two cleavages occurring in acell membrane, i.e., proteolysis at S4 that produces a Notch-β peptideand proteolysis at S3 that produces NICD determining signal transductionlevel, a mutant in which proteolysis at S3 is inhibited was prepared inthe present example and it was confirmed using this mutant that there isno change in a S4 cleavage efficiency even in the case where a S3cleavage efficiency is decreased artificially.

It has been reported that partial inhibition of S3 cleavage is caused bymutating V1744 of Notch-1 that resides on a C-terminal side with respectto a S3 cleavage site (Schroeter et al., Nature, 1998). Thus, at first,the change in a S4 cleavage activity caused by the inhibition of S3cleavage was examined. In order to efficiently detect the productsresulting from intramembranous endoproteolysis, NEXT analogues were FLAGtagged at their N-termini and myc-tagged at their C-termini. Thereafter,valine 1744 of the plasmid expressing the analogues (F-NEXT; Okochi,2002) was mutated into glycine or leucine (hereinafter these mutants arereferred to as F-NEXT V1744G and F-NEXT V1744L, respectively) (FIG. 8B).An F-NEXT expressing construct with or without S3 cleavage site mutationwas stably transfected into K293 cells constantly expressing excessivewild-type PS1 or PS1 D385N lacking a γ-secretase function. The cellsthen were metabolically labeled with ³⁵S methionine. Thereafter, newlyradiolabeled F-Nβs and NICD present in the cell sediments and thecorresponding culture media were detected by a method(IP-autoradiography) combining immunoprecipitation and radiationdosimetry performed after the separation by electrophoresis.

The cell sediments were pulsed for 30 minutes, followed byIP-autoradiography with an anti-c-myc antibody (9E10). As a result,F-NEXT expression was observed. The cells were then chased for 2 hours.As a result, NICD production caused by the degradation of F-NEXT wasobserved, whereas NICD production was inhibited significantly in theV1744G and V1744L mutants (the upper panel of FIG. 8C). These resultswere in conformity with the conventional reports. Even in the case wherethe degradation of NICD was inhibited by adding Lactacystin as aproteasome inhibitor, the amount of radiolabeled NICD measured after a2-hour chase was significantly small in the cells expressing V1744G andV1744L mutants. The intramembranous endoproteolysis and the NICDproduction caused by this F-NEXT were not at all observed in the PS1D385N expressing cells (the lower panel of FIG. 8C). From these results,it can be said that the proteolysis shown in the upper panel of FIG. 8Cwas caused by PS/γ-secretase.

Next, the culture media after a 2-hour chase were analyzed using ananti-FLAG antibody (M2). F-Nβs secreted from the F-NEXT V1744G mutantcells and the F-NEXT V1744L mutant cells were approximately the samelevel as those secreted from the wild-type F-NEXT cells (FIG. 8D).Furthermore, F-Nβ production was not observed in the cells expressingPS1 D385N mutant. From these results, it can be said that PS/γ-secretaseaffects this cleavage.

To further support the above-described conclusions, the S3 cleavageefficiency and the S4 cleavage efficiency were calculated. The ratio ofNICD to F-NEXT analogues in the cell sediments and the ratio of F-Nβs inthe culture media to the F-NEXT analogues in the corresponding cellsediments were determined. As a result, it was confirmed that althoughthe V1744G mutant and the V1744L mutant both decrease the S3 cleavageactivity in contrast to the wild-type PS1, they do not affect the S4cleavage activity (FIG. 8E).

Example 7 Correlation Between Decrease in S3 Cleavage Efficiency andAccuracy of S4 Cleavage

In PS1 mutants that cause Alzheimer's disease, the change in accuracy ofS4 cleavage occurs as well as a decrease in S3 cleavage activity. If theS3 cleavage is a precondition for the S4 cleavage, a decrease in S3cleavage efficiency caused by a PS1 mutant should affect the accuracy ofthe S4 cleavage. Thus, in the present example, a S3 cleavage site mutantwas prepared, and it was confirmed using this mutant that the accuracyof the S4 cleavage does not change even in the case where the S3cleavage efficiency is decreased artificially.

The cause of a familial Alzheimer's disease (FAD) is considered to bethat FAD pathogenic PS mutants affect the accuracy of proteolysis byPS/γ-secretase and increase the production of Aβ42, which is elongatedAβ. Similarly, the FAD pathogenic PS mutants affect the accuracy ofNotch cleavage by PS/γ-secretase and increase the production ofelongated F-Nβ. Moreover, it has been reported that some of the PSmutants cause a decrease in S3 cleavage efficiency. Thus, the effect ofS3 mutants that cause a decrease in S3 cleavage efficiency upon theaccuracy of S4 cleavage was examined. F-Nβs contained in the culturemedia of the cells expressing wild-type F-NEXT, F-NEXT V1744G mutant, orF-NEXT V1744L mutant were immunoprecipitated with M2 agarose and thenanalyzed by MALDI-TOF MS. As a result, as shown in FIGS. 9B and 9C, themajor cleavage site of the F-NEXT V1744G mutant and the F-NEXT V1744Lmutant was between alanine 1731 and alanine 1732 as in the case of thewild type, and a pattern of several minor S4 cleavage sites locatedapart from each other were not at all affected by the mutations. Inother words, it was confirmed that mutations that cause a decrease in S3cleavage efficiency do not affect the accuracy of S4 cleavage at all.These data suggest that FAD pathogenic PS mutations indirectly affectthe accuracy of S4 cleavage.

Example 8 Effect of Decrease in S4 Cleavage Efficiency Upon S3 CleavageEfficiency

Based on the assumption that S4 cleavage site mutation may exhibit asimilar effect to that of the above-described artificially prepared S3point mutants, the effect of a decrease in S4 cleavage efficiency upon aS3 cleavage efficiency was examined using F-NEXT G1730-1733 mutant andF-NEXT L1730-1733 mutant prepared by mutating four alanine residuesaround the S4 cleavage site into glycine residues and leucine residues,respectively (FIG. 10A). As a result, out of these two S4 cleavage sitemutants, the F-NEXT L1730-1733 mutant with inhibited S4 cleavageactivity exhibited a decrease in S3 cleavage efficiency. This resultsuggests that there is a proteolytic pathway through which NICD isproduced by the S4 cleavage-dependent S3 proteolysis duringintramembranous endoproteolysis of Notch-1.

Next, analysis also was made with regard to the assumption that the S4cleavage site mutants similarly may affect the S4 cleavage. As indicatedby the triangular arrowhead in FIG. 10A, a S4 cleavage site of Notch isin the center of four sequential alanine residues. The F-NEXT G1730-1733mutant and the F-NEXT L1730-1733 mutant were prepared by mutating thesefour sequential alanine residues into glycine residues and leucineresidues, respectively. These mutants then were subjected to the samepulse-chase experiment as that performed with respect to the S3 mutants.After a 2-hour chase, radiolabeled F-Nβs in the culture media wereanalyzed. As a result, F-Nβ secretion was observed in the wild-typeF-NEXT cells and the S4 mutated F-NEXT cells (FIG. 10B). However,although there was substantially no difference in the amount of F-Nβproduction between the wild-type F-NEXT and the F-NEXT G1730-1733mutant, the amount of F-Nβ production seemed to be decreased in theF-NEXT L1730-1733 mutant as compared with the wild-type F-NEXT (FIG.10B).

Next, production of radiolabeled NICD from F-NEXT contained in thecorresponding cell sediments was analyzed. As a result, a similar levelof NICD production to that of the wild-type F-NEXT cells was observed inthe G1730-1733 mutant cells, whereas NICD production was decreased inthe L1730-1733 mutant cells as compared with the cells expressingwild-type F-NEXT (the upper panel of FIG. 10C). These data suggest thatS3 cleavage is inhibited in the L1730-1733 mutant.

In order to establish this result clearly, the S4 cleavage efficiencyand the S3 cleavage efficiency were calculated in the same manner as inFIG. 8E. As a result, out of the two S4 mutants, the G1730-1733 mutantthat hardly affected the S4 activity did not affect the S3 cleavageactivity at all (FIG. 10D). In contrast, it was confirmed that theL1730-1733 mutant with inhibited S4 cleavage activity exhibited adecrease in S3 cleavage efficiency (FIG. 10D). Furthermore, from thefacts that the PS/γ-secretase mechanism causes cleavage at both S3 andS4 and that no intermediate proteolysis product resulting from the S3cleavage in close proximity to the cell membrane and the S4 cleavage atan approximate center of the transmembrane domain was found, it isconsidered the S3 cleavage and the S4 cleavage occur substantially atthe same time. These results suggest that there is a proteolytic pathwaythrough which NICD is produced by the S4 cleavage-dependent S3proteolysis during intramembranous endoproteolysis of Notch-1.

Example 9 Correlation Between S4 Cleavage Site and Activity

Subsequently, C-termini of F-Nβ G1730-1733 and F-Nβ L1730-1733 weredetermined. The amount of F-Nβs released from F-NEXT G1730-1733substantially was equal to that released from the cell expressingwild-type F-NEXT (FIG. 1B). However, the G1730-1733 mutant did not havea S4 cleavage site between glycine 1731 and glycine 1732, as indicatedby the inverse triangles in FIG. 11A. The major S4 cleavage sites ofthis mutant shifted toward the C-terminus of four sequential glycineresidues to reside between phenylalanine 1734 and valine 1735, betweenvaline 1735 and leucine 1736, and between phenylalanine 1738 and valine1739, respectively. That is, S4 cleavage did not occur around theglycine residues, but minor cleavage sites were present apart from eachother on the N-terminal side of the four glycine residues, so that MW ofF-Nβs released from the F-NEXT G1730-1733 increased (FIG. 10B).Furthermore, as indicated by the inverse triangles in FIG. 11B, theF-NEXT L1730-1733 mutant had a major S4 cleavage site in the center offour sequential alanine residues, i.e., between leucine 1731 and leucine1732, in the similar topology to that of the wild-type F-NEXT, and aminor cleavage site was hardly observed. Moreover, MW of F-Nβs releasedfrom the F-NEXT L1730-1733 mutant decreased (FIG. 10B).

INDUSTRIAL APPLICABILITY

As specifically described above, a novel polypeptide according to thepresent invention is derived from a Notch protein. In a series ofproteolytic events of the Notch protein, the polypeptide is released toan extracellular space when NICD translocates to a nucleus as a resultof intramembranous endoproteolysis that occurs subsequent toextracellular proteolysis. By using the novel polypeptide as a marker,it is possible to detect Notch signal transduction. Also, it is possibleto detect cell differentiation, cell tumorigensis, apoptosis,Alzheimer's disease, etc., for example.

1-21. (canceled)
 22. A method for detecting a condition that exhibitsabnormal Notch signal transduction, the method comprising the steps of:detecting a level of a polypeptide having an amino acid sequenceselected from a group consisting of SEQ ID NOS: 10 to 18 expressed in asample obtained from an object; comparing the level detected in thedetecting step with a level of a corresponding polypeptide detected froma normal object; and identifying abnormality of a Notch signaltransduction when the level of the polypeptide in the sample obtainedfrom the object is either significantly higher or lower than the levelof the corresponding polypeptide of the normal object.
 23. The methodfor detecting a condition that exhibits abnormal Notch signaltransduction according claim 22, wherein the detecting step of the levelof the polypeptide is conducted by using a reagent comprising anantibody that recognizes the polypeptide.
 24. The method for detecting acondition that exhibits abnormal Notch signal transduction accordingclaim 23, wherein the antibody is at least one selected from the groupconsisting of a monoclonal antibody and a polyclonal antibody.
 25. Themethod for detecting a condition that exhibits abnormal Notch signaltransduction according claim 23, wherein the reagent further comprises alabeled antibody that recognizes the polypeptide or a labeled antibodythat recognizes as an antigen said antibody that recognizes thepolypeptide.
 26. The method for detecting a condition that exhibitsabnormal Notch signal transduction according claim 25, wherein thelabeled antibody is labeled by using a fluorescent substance, an enzyme,or a radioactive substance.
 27. The method for detecting a conditionthat exhibits abnormal Notch signal transduction according claim 22,wherein the method detects a functional disorder of presenilin.
 28. Themethod for detecting a condition that exhibits abnormal Notch signaltransduction according claim 27, wherein the functional disorder ofpresenilin is at least one selected from the group consisting ofabnormal cell differentiation, tumor, apoptosis, and Alzheimer'sdisease.
 29. The method for detecting a condition that exhibits abnormalNotch signal transduction according claim 22, wherein the object is amammal.